CANCER BIOLOGY & THERAPY 2016, VOL. 17, NO. 10, 1094–1106 http://dx.doi.org/10.1080/15384047.2016.1219820
RESEARCH PAPER
The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines Maria Cosenzaa, Monica Civalleroa, Stefania Fiorcarib, Samantha Pozzia, Luigi Marchesellia, Alessia Baria, Paola Ferric, and Stefano Sacchia a
Program of Innovative Therapies in Oncology and Haematology, Department of Diagnostic Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy; bDepartment of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy; cDepartment of Diagnostic Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy
ABSTRACT
ARTICLE HISTORY
We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou–Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, ¡9, ¡3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies.
Received 25 May 2016 Revised 12 July 2016 Accepted 29 July 2016 KEYWORDS
Lenalidomide; romidepsin; synergistic interaction; T-cell lymphoma; tumor cell death
Introduction T-cell lymphomas (TCLs) comprise 10–20% of all non-Hodgkin lymphomas (NHLs). TCLs are classified into 22 subtypes by the 2008 WHO classification based on their clinicopathologic characteristics, and they comprise a various group of hematologic disease with unfortunate prognosis.1 Ordinary treatment do not generally provide satisfactory outcomes, and the majority of patients relapse. Epigenetic processes, which modify the phenotype without changing the genotype, are often altered in cancer cells.2 Epigenetic DNA and chromatin modifications include methylation, acetylation, phosphorylation, and ubiquitination. Histone acetylation is related to activation of gene transcription, whereas deacetylation is linked with transcriptional repression.3,4 The 2 processes are catalyzed by specific enzymes, namely histone acetyltransferases (HATs) and histone deacetylases (HDACs).4 HDACs enzymatically remove the acetyl group from histones and control gene expression.4 HDAC inhibitors (HDACis), which are newly emerging cancer therapeutics,5 modify genes that control cell growth and apoptosis, trigger the generation of reactive oxygen species (ROS), regulate the MAPK pathway and induce the acetylation of cytoplasmic proteins.5,6 Clinical trials have been conducted for
HDACis for the cure of cutaneous TCL (CTCL), peripheral TCL (PTCL) and Hodgkin disease.7,8 Romidepsin, also called FK228 and IstodaxÒ , is a potent HDACi with high inhibitory activity for class I HDACs.9 Romidepsin was the first HDACi to show anti-tumor activity in patients10 and has broad biological effects: it induces apoptosis, cell cycle arrest and it alters gene expression in a diversity of tumor, including TCL10,11. HDACis can be used in combination with different cytotoxic drug or anti-angiogenesis drugs.12 Lenalidomide, also called CC-5013 and RevlimidÒ , is a second-generation immunomodulatory drug (IMiD) and thalidomide analog that has antiangiogenic, antitumorigenic, and immunomodulatory activity.13,14 The use of lenalidomide in proliferative neoplasms has increased recently due to the agent’s success in multiple myeloma (MM) and myelodysplasia (MDS). In these cancers, it alters immune homeostasis and modulates inflammation within the bone marrow microenvironment. Lenalidomide treatment is effective in patients with chronic lymphocytic leukemia (CLL),15 non-Hodgkin lymphoma,16 and CTCL.17 The rationale for using drug combinations is to obtain additive or synergistic effects, thus maximizing the total dose
CONTACT Stefano Sacchi [email protected] Department of Diagnostic, Clinical, and Public Health Medicine, Program of Innovative Therapy in Oncology and Haematology, University of Modena and Reggio Emilia, Via del Pozzo, 71, 41124 Modena, Italy Supplemental data for this article can be accessed on the publisher’s website. Published with license by Taylor & Francis Group, LLC © Maria Cosenza, Monica Civallero, Stefania Fiorcari, Samantha Pozzi, Luigi Marcheselli, Alessia Bari, Paola Ferri, and Stefano Sacchi This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
CANCER BIOLOGY & THERAPY
intensity, enhancing anticancer activities, and increasing patient survival. Here we investigated combination therapy with 2 anticancer compounds that act through different mechanisms. The present study aimed to define the in vitro effects of romidepsin alone and in combination with low-dose lenalidomide in TCL cell lines and to investigate whether combination treatment could modulate apoptosis and cell viability.
Results Romidepsin and lenalidomide as single agents Romidepsin potently inhibited cell viability in both cell lines in a time- and dose-dependent manner. The IC50 ranged from 0.038 to 6.36 nM for Hut-78 cells and from 0.44 to 3.87 for Karpas-299 cells (Table 1). Important inhibition of cell vitality was evident after 48 h of incubation with romidepsin by MTT assay (Fig. 1A). Treatment with lenalidomide slightly inhibited cell viability even after 72 h of treatment but did not reach the IC50 (Fig. 1B). Drug combination shows synergistic effects in Hut-78 cells and additive effects in Karpas-299 cells Cell viability decreased significantly after treatment with both drugs compared with treatment with each drug alone. Indeed, treatment with both romidepsin and lenalidomide showed synergistic effects in Hut-78 cells, with a CI
RESEARCH PAPER
The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines Maria Cosenzaa, Monica Civalleroa, Stefania Fiorcarib, Samantha Pozzia, Luigi Marchesellia, Alessia Baria, Paola Ferric, and Stefano Sacchia a
Program of Innovative Therapies in Oncology and Haematology, Department of Diagnostic Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy; bDepartment of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy; cDepartment of Diagnostic Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy
ABSTRACT
ARTICLE HISTORY
We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou–Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, ¡9, ¡3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies.
Received 25 May 2016 Revised 12 July 2016 Accepted 29 July 2016 KEYWORDS
Lenalidomide; romidepsin; synergistic interaction; T-cell lymphoma; tumor cell death
Introduction T-cell lymphomas (TCLs) comprise 10–20% of all non-Hodgkin lymphomas (NHLs). TCLs are classified into 22 subtypes by the 2008 WHO classification based on their clinicopathologic characteristics, and they comprise a various group of hematologic disease with unfortunate prognosis.1 Ordinary treatment do not generally provide satisfactory outcomes, and the majority of patients relapse. Epigenetic processes, which modify the phenotype without changing the genotype, are often altered in cancer cells.2 Epigenetic DNA and chromatin modifications include methylation, acetylation, phosphorylation, and ubiquitination. Histone acetylation is related to activation of gene transcription, whereas deacetylation is linked with transcriptional repression.3,4 The 2 processes are catalyzed by specific enzymes, namely histone acetyltransferases (HATs) and histone deacetylases (HDACs).4 HDACs enzymatically remove the acetyl group from histones and control gene expression.4 HDAC inhibitors (HDACis), which are newly emerging cancer therapeutics,5 modify genes that control cell growth and apoptosis, trigger the generation of reactive oxygen species (ROS), regulate the MAPK pathway and induce the acetylation of cytoplasmic proteins.5,6 Clinical trials have been conducted for
HDACis for the cure of cutaneous TCL (CTCL), peripheral TCL (PTCL) and Hodgkin disease.7,8 Romidepsin, also called FK228 and IstodaxÒ , is a potent HDACi with high inhibitory activity for class I HDACs.9 Romidepsin was the first HDACi to show anti-tumor activity in patients10 and has broad biological effects: it induces apoptosis, cell cycle arrest and it alters gene expression in a diversity of tumor, including TCL10,11. HDACis can be used in combination with different cytotoxic drug or anti-angiogenesis drugs.12 Lenalidomide, also called CC-5013 and RevlimidÒ , is a second-generation immunomodulatory drug (IMiD) and thalidomide analog that has antiangiogenic, antitumorigenic, and immunomodulatory activity.13,14 The use of lenalidomide in proliferative neoplasms has increased recently due to the agent’s success in multiple myeloma (MM) and myelodysplasia (MDS). In these cancers, it alters immune homeostasis and modulates inflammation within the bone marrow microenvironment. Lenalidomide treatment is effective in patients with chronic lymphocytic leukemia (CLL),15 non-Hodgkin lymphoma,16 and CTCL.17 The rationale for using drug combinations is to obtain additive or synergistic effects, thus maximizing the total dose
CONTACT Stefano Sacchi [email protected] Department of Diagnostic, Clinical, and Public Health Medicine, Program of Innovative Therapy in Oncology and Haematology, University of Modena and Reggio Emilia, Via del Pozzo, 71, 41124 Modena, Italy Supplemental data for this article can be accessed on the publisher’s website. Published with license by Taylor & Francis Group, LLC © Maria Cosenza, Monica Civallero, Stefania Fiorcari, Samantha Pozzi, Luigi Marcheselli, Alessia Bari, Paola Ferri, and Stefano Sacchi This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
CANCER BIOLOGY & THERAPY
intensity, enhancing anticancer activities, and increasing patient survival. Here we investigated combination therapy with 2 anticancer compounds that act through different mechanisms. The present study aimed to define the in vitro effects of romidepsin alone and in combination with low-dose lenalidomide in TCL cell lines and to investigate whether combination treatment could modulate apoptosis and cell viability.
Results Romidepsin and lenalidomide as single agents Romidepsin potently inhibited cell viability in both cell lines in a time- and dose-dependent manner. The IC50 ranged from 0.038 to 6.36 nM for Hut-78 cells and from 0.44 to 3.87 for Karpas-299 cells (Table 1). Important inhibition of cell vitality was evident after 48 h of incubation with romidepsin by MTT assay (Fig. 1A). Treatment with lenalidomide slightly inhibited cell viability even after 72 h of treatment but did not reach the IC50 (Fig. 1B). Drug combination shows synergistic effects in Hut-78 cells and additive effects in Karpas-299 cells Cell viability decreased significantly after treatment with both drugs compared with treatment with each drug alone. Indeed, treatment with both romidepsin and lenalidomide showed synergistic effects in Hut-78 cells, with a CI
